1,8-ANS and bis-ANS (A47, B153; Other Nonpolar and Amphiphilic Probes—Section 13.5) have proven to be sensitive probes for partially folded intermediates in protein-folding pathways. These applications take advantage of the strong fluorescence enhancement exhibited by these amphiphilic dyes when their exposure to water is lowered (Figure 1, Figure 2). Consequently, fluorescence of ANS increases substantially when proteins to which it is bound undergo transitions from unfolded to fully or partially folded states that provide shielding from water. Molten globule intermediates are characterized by particularly high ANS fluorescence intensities due to the exposure of hydrophobic core regions that are inaccessible to the dye in the native structure. Binding of 1,8-ANS and bis-ANS to proteins is noncovalent and involves a combination of electrostatic and hydrophobic modes. Some investigators have noted that the dye-binding event itself may induce protein conformational changes, indicating the advisability of correlating ANS fluorescence measurements with data obtained using other physical techniques. In particular, high-resolution structural analysis of an ANS–protein complex by X-ray crystallography has demonstrated the occurrence of local rearrangements of the protein structure to accommodate the dye.

Figure 1. Fluorescence emission spectra of equal concentrations of 1,8-ANS (A47) in ethanol:water mixtures. The labels adjacent to each curve indicate the percentage of ethanol in the solvent mixture.

Figure 2. Fluorescence enhancement of 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid, A47) upon binding to protein. The image shows aqueous solutions of 1,8-ANS excited by ultraviolet light. Addition of protein (bovine serum albumin) to the solution in the cuvette on the left results in intense blue fluorescence. In comparison, the fluorescence of uncomplexed free dye in the cuvette on the right is negligible.